title: Simultaneous recovery of dual pathways for ammonia metabolism do not improve further detoxification of ammonia in HepG2 cells.
abstract: BACKGROUND
  Key enzyme deficiency in the dual-pathway of ammonia metabolism leads to low detoxification capacity of HepG2 cells. Previously, we established a HepG2/AFhGS cell line with overexpression of human glutamine synthetase (hGS) in pathway 1 and a HepG2/(hArgI+hOTC)4 cell line with overexpression of human arginase I (hArgI) and human ornithine transcarbamylase (hOTC) in pathway 2. The present study aimed to investigate whether simultaneous recovery of the two pathways contributes to the further improvement of ammonia detoxification in HepG2 cells.
  
  
  METHODS
  We adopted a recombinant retrovirus carrying the hGS gene to infect HepG2/(hArgI+hOTC)4 cells and selected a new recombinant HepG2 cell line. The capacities of ammonia tolerance and detoxification in cells were detected by biochemical methods. Cell cycle PCR chip was used to assess the changes of gene expression.
  
  
  RESULTS
  Introducing hGS into HepG2/(hArgI+hOTC)4 cells did not lead to hGS overexpression, but inhibited hArgI expression. The levels of synthetic glutamine and urea in HepG2/(hArgI+hOTC+AFhGS)1 cells were significantly lower than those in HepG2/(hArgI+hOTC)4 cells when cultured in the medium with 10 and 15 mmol/L glutamate (Glu) and with 60 and 180 mmol/L NH4Cl, respectively. In addition, the comparison of different cell growth showed that HepG2/AFhGS cells significantly lagged behind the other cells by the 5th and 7th day, indicating that introduction of hGS impedes HepG2 cell proliferation. Analysis of the mechanism suggested that the decreased expression of BCL2 played an important role.
  
  
  CONCLUSIONS
  This study demonstrated that the recovery of two ammonia metabolic pathways in HepG2 cells is not helpful in increasing ammonia metabolism. The reinforcement of the pathway of urea metabolism is more important and valuable in improving the ammonia metabolism capacity in HepG2 cells.
authors.affiliation: Fujian Institute of Hepatobiliary Surgery, Fujian Medical University Union Hospital, Fuzhou 350001, China. fztnh@sina.com.
authors.forename: Fei-Yuan
authors.initials: FY
authors.lastname: Zhang
authors.forename: Nan-Hong
authors.initials: NH
authors.lastname: Tang
authors.forename: Xiao-Qian
authors.initials: XQ
authors.lastname: Wang
authors.forename: Xiu-Jin
authors.initials: XJ
authors.lastname: Li
authors.forename: Yan-Ling
authors.initials: YL
authors.lastname: Chen
mesh_terms: D000641:Ammonia / Q000378:metabolism*; D001119:Arginase / Q000235:genetics / Q000378:metabolism; D001711:Biotransformation; D002453:Cell Cycle; D049109:Cell Proliferation; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D005974:Glutamate-Ammonia Ligase / Q000235:genetics / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D009954:Ornithine Carbamoyltransferase / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014508:Urea / Q000378:metabolism
chemical_list: D000641:Ammonia; D014508:Urea; D009954:Ornithine Carbamoyltransferase; C574619:ARG1 protein, human; D001119:Arginase; D005974:Glutamate-Ammonia Ligase